Craniofacial and Skeletal Diseases Branch Matrix Metalloproteinase Unit seeks to understand the biological roles of Matrix Metalloproteinases (MMPs) and their inhibitors in development, homeostasis and disease with specific emphasis on the role of MMPs in skeletal and peri-skeletal tissues. The MMP family is considered important in tissue remodeling and our studies using in vitro as well as mouse genetic approaches in this field have demonstrated that MT1-MMP, a membrane-bound MMP, is essential for the timely removal of type I and type II collagen matrices. We have maintained efforts to specifically identify substrates of MMPs as well as their regulation in disease processes. MT1-MMP IS REQUIRED FOR OSTEOCYTE PROCESSES (40% Effort). We have previously demonstrated that osteogenic cells have an obligate requirement for MT1-MMP to sustain bone formation and remodel the collagen rich osteogenic periskeletal tissues. We posed the question whether or not this requirement is also shared by osteocytes, which in general are considered relatively quiescent osteogenic cells entombed in mineralized matrix of the bone. Surprisingly, osteocytes, like bone forming cells, expressed copious quantities of MT1-MMP message. In addition, it was demonstrated that specific collagenase cleavage products were conspicuously associated with the osteocyte process network and that MT1-MMP also localized there. In MT1-MMP deficient mice, osteocyte processes residing in canaliculae of bone, were conspicuously absent. This finding suggests that osteocytogenesis is an active invasive process required to maintain space for the cell process in an environment which by default will mineralize and restrain cells. By maintaining a constantly low level of matrix synthesis and dissolution, we propose that osteocytes and their cell processes can evade encroachment that would be caused by default mineralization of unremodeled osteoid matrix. As such, osteocytogenesis is not merely a passive sequestration of cells in a mineralized matrix, but rather an active invasive process ensuring maintenance of the network of osteocyte processes. We demonstrated that this process is selectively MT1-MMP specific, as mice deficient for the collagenolytic MMP-13 have abundant osteocyte processes MT-MMP EXPRESSION PATTERN IN DYNAMICALLY REMODELING TISSUES (40% Effort) In an effort to further our understanding of the biological roles of several membrane type (MT) MMPs in dynamic tissue remodeling of pathological and non-pathological nature, we undertook a thorough expression analysis of healthy and diseased mammary gland and mapped the expression of MT1, MT2, MT3, and MT4-MMP. We utilized mammary gland because initial development, lactation, involution and reconstitution of the gland are associated with substantial tissue remodeling of both stromal and epithelial compartments. We documented that MT1, 2 and 3 were expressed in both anabolic and catabolic tissue remodeling processes whereas steady state lactation, which is associated with little or no structural remodeling suppressed MMP expression. Consistently we failed to detect MT4-MMP. The analysis moreover demonstrated that MT1-MMP and MT3-MMP were expressed in the stromal compartment with distinctly different patterns, although there was partial overlap between the two patterns. In contrast, MT2-MMP was stringently restricted to the epithelial compartment. Moreover we demonstrated that the pattern of expression observed in the normal mammary gland was mirrored in a genetic model of adenocarcinoma. Significantly, MT1-MMP was substantially upregulated in the tumor-associated stroma. In aggregate, these results provide evidence that despite organ co-expression, the MT-MMPs are confined to distinct tissue compartments from which some of their substrate specificities and biological functions may be inferred. EXTRACELLULAR AND INTRACELLULAR COLLAGEN METABOLISM (20% Effort) During analysis of the MT1-MMP deficient mice, we encountered connective tissue cells with conspicuous intracellular collagen inclusions. We have characterized in detail that this collagen is accumulating in the phagosomes whereas the phagolysosomal compartment is devoid of collagen. Based on the literature describing the phagocytic collagen metabolism, we inferred that loss of pericellular collagenase activity is causing a switch in metabolic pathway, which lead to an overload of the phagocytic uptake. uPARAP/endo180 is a recently identified surface receptor for collagen, which is essential for collagen uptake and routing to the phagocytic pathway. In an effort to elucidate the combined significance of extracellular and intracellular collagen processing, we have generated mice double deficient for MT1-MMP and uPARAP. Our analysis of these animals demonstrate that they suffer from diminished bone formation over and above what is observed in MT1-MMP single deficient mice. Moreover the combined deficiency is associated with uniform peri-natal death demonstrating an uncompensated loss of collagen processing, which is required for proper bone formation and growth. Taken together these observations highlight the important residual capacity of the cell for processing collagen by means of intracellular uptake in cases when the matrix load overwhelms the peri-cellular proteolysis. ON-GOING STUDIES The maintenance of a permanent hyaline cartilage is essential for joint homeostasis, however, we observe in our analysis of MT1-MMP deficient animals that the interface between cartilage and bone is a site critically dependent on timely remodeling. Deficiency in this process has proved to significantly impair growth and generates a severe arthropathy. This highlights the significance of MT1-MMP in cartilage degradation in the process of growth, but also suggests that MT1-MMP may be a significant proteolytic enzyme in pathological joint destruction. In an effort to understand this mechanism further, in our current studies, we have directed MT1-MMP expression into chondrocytes. Surprisingly we observe none of the expected effects on cartilage degradation, suggesting that cartilage dissolution is tightly regulated by both permissive extracellular matrices, as well as inhibitors in the local environment.